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Cold adaptation of ...
Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.
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- Lönn, Anna (författare)
- Lund University,Lunds universitet,Teknisk mikrobiologi,Centrum för tillämpade biovetenskaper,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Applied Microbiology,Center for Applied Life Sciences,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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- Gárdonyi, Márk (författare)
- Lund University,Lunds universitet,Teknisk mikrobiologi,Centrum för tillämpade biovetenskaper,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Applied Microbiology,Center for Applied Life Sciences,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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van Zyl, Willem (författare)
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- Hahn-Hägerdal, Bärbel (författare)
- Lund University,Lunds universitet,Teknisk mikrobiologi,Centrum för tillämpade biovetenskaper,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Applied Microbiology,Center for Applied Life Sciences,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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Otero, Ricardo Cordero (författare)
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(creator_code:org_t)
- 2003-10-30
- 2002
- Engelska.
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 269:1, s. 157-163
- Relaterad länk:
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http://dx.doi.org/10...
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https://febs.onlinel...
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https://lup.lub.lu.s...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.
Ämnesord
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology (hsv//eng)
Nyckelord
- Hydrogen-Ion Concentration
- Kinetics
- Magnesium : pharmacology
- Manganese : pharmacology
- Mutagenesis
- Polymerase Chain Reaction
- Support Non-U.S. Gov't
- Xylitol : pharmacology
- Thermus thermophilus : enzymology
- Enzyme Stability
- Cold
- Aldose-Ketose Isomerases : chemistry : genetics : metabolism
Publikations- och innehållstyp
- art (ämneskategori)
- ref (ämneskategori)
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